Orientation selective DBS of entorhinal cortex and medial septal nucleus modulates activity of rat brain areas involved in memory and cognition

The recently introduced orientation selective deep brain stimulation (OS-DBS) technique freely controls the direction of the electric field’s spatial gradient by using multiple contacts with independent current sources within a multielectrode array. The goal of OS-DBS is to align the electrical field along the axonal track of interest passing through the stimulation site. Here we utilized OS-DBS with a planar 3-channel electrode for stimulating the rat entorhinal cortex (EC) and medial septal nucleus (MSN), two promising areas for DBS treatment of Alzheimer’s disease. The brain responses to OS-DBS were monitored by whole brain functional magnetic resonance imaging (fMRI) at 9.4 T with Multi-Band Sweep Imaging with Fourier Transformation (MB-SWIFT). Varying the in-plane OS-DBS stimulation angle in the EC resulted in activity modulation of multiple downstream brain areas involved in memory and cognition. Contrary to that, no angle dependence of brain activations was observed when stimulating the MSN, consistent with predictions based on the electrode configuration and on the main axonal directions of the targets derived from diffusion MRI tractography and histology. We conclude that tuning the OS-DBS stimulation angle modulates the activation of brain areas relevant to Alzheimer’s disease, thus holding great promise in the DBS treatment of the disease.

The recently introduced orientation selective deep brain stimulation (OS-DBS) technique freely controls the direction of the electric field's spatial gradient by using multiple contacts with independent current sources within a multielectrode array. The goal of OS-DBS is to align the electrical field along the axonal track of interest passing through the stimulation site. Here we utilized OS-DBS with a planar 3-channel electrode for stimulating the rat entorhinal cortex (EC) and medial septal nucleus (MSN), two promising areas for DBS treatment of Alzheimer's disease. The brain responses to OS-DBS were monitored by whole brain functional magnetic resonance imaging (fMRI) at 9.4 T with Multi-Band Sweep Imaging with Fourier Transformation (MB-SWIFT). Varying the in-plane OS-DBS stimulation angle in the EC resulted in activity modulation of multiple downstream brain areas involved in memory and cognition. Contrary to that, no angle dependence of brain activations was observed when stimulating the MSN, consistent with predictions based on the electrode configuration and on the main axonal directions of the targets derived from diffusion MRI tractography and histology. We conclude that tuning the OS-DBS stimulation angle modulates the activation of brain areas relevant to Alzheimer's disease, thus holding great promise in the DBS treatment of the disease.
Although Deep Brain Stimulation (DBS) is an established therapeutic modality for the treatment of movement disorders, its utility for Alzheimer's disease is also becoming increasingly evident [1][2][3] . The entorhinal cortex (EC) 4 , fornix 5 , nucleus basalis of Meynert (NBM) 6 , anterior nucleus of the thalamus 7 and medial septal nucleus (MSN) 8 are promising DBS targets for improving dysfunctional memory and other cognitive functions. Putative mechanisms of actions include alteration of neuronal firing patterns, increase communication across several brain regions, amplification of synaptic plasticity 9 and induction of neurogenesis 7 . In particular, DBS of the EC 10 and MSN 8 induces neurogenesis by promoting neural stem proliferation in the dentate gyrus subgranular zone of the hippocampus (HC). After DBS of the EC in mice, the newly born nerve cells can integrate into the neural circuitry of the dentate gyrus, survive long term, and there is a relationship between stimulation-induced promotion of neurogenesis, differentiation of neural stem cells to mature dentate granule cells, and enhanced spatial memory 10 . Experimental work in rodent models further indicated that fornix DBS may lead to improved memory via elevation of acetylcholine release in HC 11 and induction of growth factors 12  www.nature.com/scientificreports/ Several functional features of the EC and MSN corroborate their use as potential targets for the treatment of Alzheimer's disease. In particular, the EC provides the major cortical input to the HC, and transgenic mice in which these inputs are inhibited display impaired temporal association memory 13 . On the other hand, MSN acts as a "pacemaker" in regulating hippocampal theta oscillations [14][15][16] and its stimulation prior to a spatial working memory task has been shown to enhance hippocampal theta activity and improve spatial working memory 17 . In brain slices, a cholinergic agonist amplifies the theta rhythm and increases the sensitivity of HC synapses to undergo long-term potentiation or depression 18 . Thus, MSN stimulation may improve memory encoding by strengthening the theta rhythm through augmenting the cholinergic input to the HC. Moreover, in Alzheimer's disease mouse model, optogenetic stimulation of medial septal parvalbumin neurons could restore the hippocampal gamma oscillations despite significant plaque deposition 19 . Beyond Alzheimer's disease, MSN stimulation can be a valuable "proxy intervention" 20 also for epilepsy, since structures such as the hippocampus and entorhinal cortex are often involved in the disease. Both optogenetic and electrical stimulation of the MSN have indeed been shown to reduce seizures in HC and rescue memory function in epileptic rats [20][21][22] . DBS of the MSN thus yields great promise for translation in patients with temporal lobe epilepsy who have memory deficits.
Initial clinical trials have been conducted to assess the efficacy of DBS in patients with Alzheimer's disease 23 . In a pilot study of six patients with mild to moderate Alzheimer's disease, improved cognitive performance and enhanced 18 F-glucose uptake in the brain were observed following stimulation of the NBM 24 . Other studies have suggested that targeting the fornix leads to improved cognition 25 , enhanced cortical and hippocampal 18 F-glucose uptake 26 , and increased hippocampal volumes 27 . However, a subsequent multicenter phase II trial on fornix stimulation found no significant differences between the DBS and control groups with respect to primary cognitive outcomes 28,29 . This lack of DBS efficacy may be at least partially attributed to inadequate target selectivity during DBS.
To advance the spatial selectivity of neuronal modulation with DBS, our group pioneered a novel orientationselective strategy for DBS, entitled OS-DBS 30 . This strategy entails that, by using multiple contacts with independent current sources within a multielectrode array, the electric field can be oriented along any desired orientation in space, such that axons parallel to the electric field spatial gradients are preferentially activated. The OS-DBS technique has been successfully used in rodents with a planar three-channel electrode to attain in-plane reorientation of the primary direction of the electrical field in stimulation sites encompassing the corpus callosum 30 , the infralimbic cortex (IL) 31 , and the subthalamic nucleus (STN) 32 . Additionally, we have demonstrated that OS-DBS shows promise to significantly improve the clinical outcomes of DBS therapy when using commercially available DBS leads in clinical settings 33 .
In this study, OS-DBS with a three-channel electrode was utilized for stimulation of the rat EC and MSN with the goal of modulating the activation of brain networks connected to the stimulation sites. The stimulation effects were monitored by means of whole brain fMRI with Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT). Such imaging modality operates with virtually no echo time and large bandwidth (BW), and thus minimizes artefacts from implanted electrodes and motion 34,35 . The effect of OS-DBS on the fMRI maps was evaluated in single subjects and at group level. Moreover, a region of interest (ROI) analysis was performed to quantify fMRI activation strength in downstream areas critical to Alzheimer's disease, and/or connected to the targets. For the EC OS-DBS, the primary ROIs included dorsal and ventral hippocampus (DHC and VHC, respectively), subiculum (Sub), as well as other areas connected to the EC such as perirhinal cortex (PrC), piriform cortex (Pir), amygdala (Amg) and insula (Ins). For the MSN case, the primary ROIs included the DHC and VHC, Sub, and other areas connected to the MSN such as the interpeduncular nucleus (IP), the lateral hypothalamus (LH), the mammillary bodies (MM) and supramamillary nuclei (SuM). Finally, in order to substantiate the fMRI findings during OS-DBS, we characterized the main axonal orientations of the EC and MSN by diffusion MRI tractography and histological assessments.

Results
All fMRI sessions conducted during OS-DBS in both EC and MSN ( Fig. 1) were successfully completed and provided data of sufficient quality for subsequent fMRI analyses. However, in two rats out of 12 undergoing OS-DBS in the EC, the electrode was determined to be out of target based on the estimated dorsal-ventral (DV) location of the electrode tip obtained by overlapping the MRI images with the rat brain atlas. Therefore, these rats were excluded from further analyses. In the remaining 10 rats of the EC group, the electrode DV coordinates were 8.1 ± 0.2 mm from dura, and the delivered current amplitudes were 1.7 ± 0.4 mA ( Supplementary Fig. 1). For the 8 rats of the MSN group, the DV coordinates were 6.0 ± 0.2 mm, and the current amplitudes 1.0 ± 0.3 mA ( Supplementary Fig. 2).
For the EC stimulation, we first conducted preliminary fMRI studies in one rat (not shown) at various frequencies (namely 20 Hz, 70 Hz, and 130 Hz) and found that robust fMRI response in the hippocampus was detected at 20 Hz, which was thus chosen for the rest of the study. This was also the frequency that most effectively activated HC with perforant path stimulation in an earlier study 37 . OS-DBS of the EC at 20 Hz robustly activated numerous downstream brain regions. The activated areas were mainly located on the right hemisphere in accordance with the stimulation side. Varying the stimulation angle led to brain network modulations which could be appreciated in single subjects ( Supplementary Fig. 3), as well as at group level (Fig. 2) despite a substantial inter-subject variability. No group main effects were seen at − 90°, although 4 out of 10 rats exhibited brain activation at this angle. Significant group main effects in VHC, Sub, Amg, and PrC occurred at 0° and − 45°, while group main effects in Pir and Ins generally occurred at 180°. In addition, a significant main effect was found in VHC at − 135°. Beside activation in the primary ROIs, the main effect maps revealed significant activations at various angles also in other brain areas, including caudate-putamen (CP), diagonal band (DB), hypothalamus . While modulations were seen also for the DHC, Sub, and PrC, they did not reach statistical significance.
The typical 130 Hz frequency used in DBS clinical settings 23,25 was found to provide robust HC response in case of MSN stimulation, and was selected for the subsequent experiments. OS-DBS of the MSN at 130 Hz activated numerous brain regions ( Fig. 4 and Supplementary Fig. 4), including the primary ROIs, DHC and VHC, Sub, LH, MM, SuM and IP, and additional areas, Amg, DB, IL/PL, LS, MS, NA, SN, VP, and ventral tegmental (VT) area. These were mainly right lateralized in accordance with the verified right placement bias of the stimulation electrodes. However, no angle-dependence of the brain activity patterns were observed, neither at voxel-level in single subject ( Supplementary Fig. 4) and in the group (Fig. 4), or at ROI-level (Fig. 5).
The orientations of myelinated axons in the EC and MSN were assessed with histology (Fig. 6a,c) and diffusion MRI tractography (Fig. 6b,d,e). For more superficial EC layers, the histology shows that the axons are too sparse to identify a main orientation, while the deeper myelin sections exhibited main axon orientations ranging from about − 45° to 180°. Tractography shows complex axon distributions in the implantation sites, consistent with the observation that multiple angles at times provided comparably strong fMRI clusters in individual rats ( Supplementary Fig. 3). Despite the observed complex axon distributions in each rat, − 45° appeared to be one of the main axon orientations around the target position in most rats ( Supplementary Fig. 1). Notably, OS-DBS stimulations at − 45° provided distinctively bigger fMRI clusters in connected areas to the EC, including HC, Sub, Amg, and PrC (Fig. 2).
For the MSN, the main axonal orientation was dorsal-ventral, i.e., along the electrode itself. Such observation explains the lack of orientation selectivity observed for OS-DBS of MSN, as in fact the reorientation of the stimulation angle was achieved on the plane encompassing the tips of the 3-channel electrode perpendicular to the major fiber orientation.  www.nature.com/scientificreports/

Discussion
In this work, we demonstrated that varying the stimulation angle in OS-DBS can be exploited to modulate the activity of the networks connected to the EC and MSN. Maximal fMRI responses of downstream areas to the EC generally occurred at stimulation angles of − 45°, which was one of the main axonal directions of the EC nerve fiber efferents observed in deep horizontal sections (i.e., further from the skull, Fig. 6a), and 180°. The ventral region of the hippocampus, which is closer to the EC in rodents, was primarily activated during OS-DBS of the EC, and showed a stimulation angle effect. On the other hand, for the MSN group, the fMRI responses in HC and brain areas connected to the MSN did not vary significantly between different stimulation angles with the planar 3-channel electrode used here. Being a midline region, MSN is a particularly promising target for stimulation. Brain activations during MSN stimulation were indeed very robust and covered the entire HC, in agreement with findings that MSN optogenetic stimulation modulates electrical activity in the rostral to caudal extent of the HC 21 . Note that, in these data, activations were more prominent in one hemisphere because the electrodes were often slightly offset to the right side of the MSN. This study shows the feasibility of OS-DBS for stimulating areas relevant to Alzheimer's disease, setting the framework for further technological developments and applications of multielectrode arrays to substantiate the potential of our findings as a direction for treatment therapy in Alzheimer's disease. The flexibility offered by OS-DBS in enhancing the activation of a specific circuitry by targeting the axonal directions of interest within the stimulation site may be beneficial to reduce stimulation-related side effects while enhancing therapeutic effects in chronic stimulation settings. Although statistically significant group-level modulatory effects induced by the stimulation angle were observed in our group of 10 rats implanted in the EC, large variability of activation patterns as a function of the stimulation angle was also observed. As the EC is comprised of five cortical layers 38 , with different layers projecting differently to the HC, the location of the electrode relative to such layers has the potential to significantly affect the outcomes of DBS. Specifically, neurons in layer II project to the dentate gyrus and CA3, while neurons in layer III have projections to CA1 and subiculum 38 . The deep layers, especially layer V, receive projections from the HC 39 , and project to EC superficial layers and extrahippocampal brain structures 38 . Moreover, some axons project to extrahippocampal brain structures like amygdala, some axons project to the layer II of EC then to CA1 and dentate gyrus, some axons project to layer III then to CA3. Based on such considerations, it is thus conceivable that OS-DBS leads to somewhat different activation patterns when stimulating the deeper layer as compared to the superficial layers, a prediction that may explain the large inter-subject variability of modulatory effects induced by OS-DBS as observed in our study. In addition, it should be kept in mind that the large electrode size could lead to some axons in deeper layers to be stimulated when stimulating the superficial www.nature.com/scientificreports/ layers. Importantly, it should be noted that the convention used for assigning "superficial" and "deep" horizontal myelin staining sections in Fig. 6a generally follows the convention of superficial and deep cortical layers 38 , but a precise delineation of the cortical layers as defined in 38 for each horizontal section cannot be achieved in the absence of a complete 3D structure determination of the EC. Therefore, a one-to-one assignment of electrode location in each rat within the exact cortical layer cannot be robustly accomplished. The complex nature of the EC architecture was confirmed by the complementary information obtained from the tractograms (Fig. 6b), which showed a range of orientations of the major tracts according to the slice position from deep to superficial layers. Worth noting is that histology provides information only about myelinated axons, while diffusion MRI tractograms provide information about all the structures affecting water molecule movement. www.nature.com/scientificreports/ Absence of brain activations was observed at − 90° in the group analysis and, at single subject level, in 6 out of 10 rats implanted in EC. This was likely because at − 90° the negative pole of the stimulation vector was quite close to the edge of the brain where the density of the axons is lower. OS-DBS at − 45° resulted in large spread of activation both in the group analysis and in the majority of rats at single subject level, which was in agreement with main axonal orientations in the deep layers indicated in Fig. 6. At 135°, which is opposite to the − 45° orientation, the fMRI response was minimal. This could be as a result of partial anodic block generated by the anodic lobe of the dipole stimulation 40 .
Several other brain structures also exhibited strong fMRI responses when stimulating the EC, including the amygdala and IL/PL, which are involved in memory consolidation 41 . The amygdala receives strong EC projections, mainly from lateral entorhinal cortex (LEC) [42][43][44] , and further connects with the ventral (temporal) CA1 subdivision of the HC, and perirhinal cortex 45 . The IL/PL also receives major projections from LEC 46 . Further, we observed activation in the nucleus accumbens, which receives direct EC projections 47 . In addition, strong activation was seen in piriform cortex and insula, both of which receive direct projections from LEC 38 .
OS-DBS of the MSN elicited strong fMRI responses in the dorsal and ventral HC, subiculum, amygdala, and medial frontal cortex (IL/PL). In addition, MSN stimulation activated the mamillary and supramamillary nuclei that receive hippocampal projections through the fornix. For MSN, the main orientation of myelinated axons was dorsal-ventral (Fig. 6d). Therefore, varying the main electrode field direction in an axial plane did not result in significant differences in activation patterns (Fig. 4, Supplementary Fig. 4). Future studies are thus required for achieving OS-DBS along a dorsal-ventral orientation, for which electrodes with minimally four independently driven channels distributed in 3D rather than on a plane are required for re-orienting the electric field gradients in space.
The MRI findings most likely reflect not only contributions from orientation selective stimulation of axons within the target, but also from the specific location of the effective cathode during OS-DBS. For instance, the activation of the HC could be induced because of proximity of the effective cathode to the angular bundle and subiculum, where fibers from the EC converge with primary orientations of 0° and 90° and project to the hippocampus. This conceptual framework is supported by the observation that strong HC activation occurs at 0°, although is supported to a less extent at 90° where we do not see a response as strong as at 0°. The substrates that mediate the activation of other structures at angles of 180° and -135° are less obvious, but at these angles, the stimulation is directed towards the entorhinal cortex, including its deep layers that project to cortical areas including those activated at these angles. www.nature.com/scientificreports/ Several limitations of the study should be noted. First, the sample size of the study was relatively small (10 animals for EC implantation and 8 for MSN implantation). To mitigate this limitation, we used strict threshold criteria in the generation of group-based activation maps, and further document the individual single subject results. Moreover, the relatively large size of the 3-channel electrode, along with the high stimulus intensity used in these acute DBS study, imply that different OS-DBS angles may stimulate surrounding areas of the selected targets thus leading to widespread activations beyond those of the targeted circuitry. However, on the other hand, a small contact size could lead to tissue damage because of the high energy deposition per unit time. Higher electrode counts and different electrode configuration are also desired to achieve re-orientation of the electrical field in space rather than on a plane like that exemplified in the present study. Despite the use of the stereotactic system and despite all efforts had been allocated to ensure precise implantation, implantation inaccuracies can still take place, potentially introducing variability in the activation patterns even within the same group of animals. The electrodes may also be inserted into the tissue in slightly twisted manner, so the actual determination of the stimulation angles may be compromised across the animals thus preventing accurate group analyses as a function of the stimulation angle. Moreover, the characterization of fiber orientations based on tractography and myelin staining is highly informative, but it has also limitations. We can extract fiber orientation from tractograms reconstructed at 150-µm thick slice. As a grey matter area, the presence of fiber bundles in EC is low as observed in myelin staining, and the tractograms reflect all orientations of the structures contained in that relative thick slab of tissue, including myelinated, non-myelinated axons or dendrites. For a more accurate estimation of the angular differences between OS-DBS experiments, higher resolution diffusion MRI, other histological approaches, e.g. 3-dimensional confocal microscopy, and determination of electrode locations should ideally be conducted in each individual animal, rather than using a reference brain as it was done in the present study. Yet, the a priori knowledge of axon orientations is helpful for narrowing the parameter space of stimulations angles and for the validation purposes, but it is not intrinsically required for exploiting the OS-DBS framework. Indeed, the flexibility offered by OS-DBS to vary the stimulation angle, together with the opportunity to measure whole-brain activity with fMRI during OS-DBS, allows to maximize in real-time the activation of connected www.nature.com/scientificreports/ downstream brain areas also when the direction of axons is not precisely known in the individual subject, and even in presence of minor electrode placement inaccuracies. The 1-2 mA current used in this study, higher than typically utilized clinically, was motivated by the need of achieving robust fMRI responses. It should be noted that anesthesia itself most likely challenges the efficacy of electrical stimulation for inducing fMRI responses. Moreover, the implantation of the DBS electrode results in acute edema around and along the lead, causing a decrease in the amount of current spreading into the tissue and a decrease in extracellular signal amplitude 48 that may reflect in smaller fMRI contrast. Worth noting is that the presence of edema can also compromise the selectivity of stimulation given the current spreading into the surrounding areas beyond the target. Notably, our preliminary results in awake animals with chronically implanted DBS electrodes (unpublished), where anesthesia does not play a role and the edema around the electrode is supposed to be resolved, suggest that the level of current needed for achieving detectable fMRI responses is up to one order of magnitude lower than in anesthetized rats. Overall, the high currents used in anesthetized animals may cause tissue damage, and even the MRI pulse sequences can induce unwanted currents in DBS leads because of RF heating of the tip of the electrode 49,50 . Histological evaluations were not conducted in this work, however visual inspection of the anatomical images at the end of the experiments suggested no tissue damage, consistent with other DBS studies in rodents when using balanced bipolar pulses with similar level of current 1-2 mA [30][31][32]34,51,52 .
The use of anesthesia in the current study allowed acquiring fMRI responses to stimulation at several angles for investigating the effects of OS-DBS. However, awake animals cannot remain comfortable in the scanner for more than ∼45 min. Therefore, OS-DBS study designs will require fewer stimulation angles and/or shorter stimulation trains to achieve tolerable scan times, despite the animal is expected to tolerate well long stimulation trains because lower currents can be used in the awake state. Worth noting, while lower currents will benefit safety and stimulus tolerance, they may potentially impair stimulus efficacy and selectivity. In fact, previous neuronal modelling results 30 showed clear OS-DBS effects with 1-2 mA currents and similar electrode design used in this study, but lower currents and different electrode configurations were not explicitly investigated, including different inter-contact distances or higher channel counts. Overall, dedicated modeling and experimental investigations will need to be undertaken to evaluate electrode designs, stimulus parameters, and MRI protocols with the goal of optimizing OS-DBS in awake animals. Finally, for EC stimulation, in the present study we used a 20 Hz stimulation frequency, which is a classic kindling paradigm for inducing epilepsy 53 . Therefore, future studies may be focused on circumventing kindling by utilizing high inhibitory frequency of stimulation, 100 Hz and higher. High frequency stimulation (HFS) is used in treating Parkinson's disease patients with DBS targeted to globus pallidus (GP) or STN. In both cases, DBS at these frequencies decrease firing of talamocortical neurons. The exact underlying neural mechanisms are still somewhat unsettled, but the general idea as reviewed by Liu and colleagues 54 is that HFS suppresses firing at the nearby neuronal somata, possibly via a depolarization block, while nearby axons are stimulated at the applied frequency. In case of basal ganglia, the axons stimulated at these high frequencies are the GABAergic axons of GP neurons, which inhibit STN or thalamic glutamatergic neurons. In case of entorhinal cortex stimulation, similarly GABAergic neurons impinging on layer 2 projection neurons may get activated. It has been shown that prolonged (> 10 min) HFS at EC does not evoke seizures in mice 10 . Future studies are also warranted in larger animal models to better resemble clinical settings.
In conclusion, we conducted the first in vivo investigations of OS-DBS of the EC and MSN in rats. Such stimulation targets are potential DBS targets in Alzheimer's disease given their involvement in memory and cognition and their association with induction of neurogenesis in the dentate gyrus of the hippocampal formation. OS-DBS of the EC modulated brain activity in the HC which is crucial for spatial and episodic memory, in the amygdala which is involved with emotional memory modulation, in the medial frontal cortex (IL/PL) involved with several cognitive functions. Strongest activation of HC, subiculum, amygdala, and perirhinal and piriform cortex was achieved when stimulating the EC at − 45° at the group level, in general agreement with the outcomes provided by histological and MRI tractography assessments. No significant dependence of the activation on OS-DBS of the MSN had been detected, which was attributed to the orthogonal orientation of axonal tracks relatively to the stimulation plane of the implanted 3-channel electrode. Further studies with multichannel electrode with higher channel count, and in larger animals using commercial electrodes, are required for substantiating the potential of OS-DBS as a treatment therapy for Alzheimer's disease. Since OS-DBS can be implemented with available commercial electrodes 33 , and fMRI can be safely used during DBS 55 , the conceptual framework of combining OS-DBS with fMRI for manipulating and monitoring brain network activity, respectively, holds great promise for translation to clinical settings.

Methods
Surgical procedures and electrode implantations. The study was carried out in compliance with the ARRIVE guidelines, and all surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota. Sprague-Dawley rats (Envigo; Madison, WI, USA; male, 252-340 g, n = 12 for EC; n = 8 for MSN) were housed in pairs in a temperature and humiditycontrolled vivarium with a 12-h light-dark cycle with ad libitum diet. Rats were initially anesthetized using isoflurane for the duration of the implantation (5% for induction, 1-3% during surgery) with O 2 /N 2 O (30%/70%) carrier gas. The respiration rate was monitored using a plastic pressure sensor during the whole study. The temperature was monitored using a rectal thermometer and maintained at 37 °C with a heating pad during the surgery, heated water circulation and heated air during MRI. After the electrode implantation, the anesthesia was changed to urethane (1.5 g/kg) with four consecutive injections 15 min apart while gradually lowering the isoflurane level to reach 0%. Urethane was used to replace isoflurane as it enables a strong fMRI response 56 and maintains normal blood gas levels in spontaneously breathing rats 57 . Each animal was placed on a stereotactic frame and a craniotomy was made by drilling through the skull on the right hemisphere. A tripolar lead com- www.nature.com/scientificreports/ posed of a twisted set of three polyimide-insulated tungsten wires (PlasticsOne, MS333T/2C-A/SP; Roanoke, VA, USA) with tip-contact diameters of 127 µm, and including the insulation layer 157 µm (total diameter of three electrode bundle ~ 350 µm), were implanted in the right EC and MSN. For EC, the targeted coordinates were: anterior-posterior (AP) = − 6.85 mm, medio-lateral (ML) = − 5 mm, and dorsal-ventral (DV) = 8 mm; for MSN, they were AP = + 0.1 mm, ML = + 0.4 mm, and DV = 5.8 mm based on the rat atlas 36 . Three to four drops of 2% lidocaine were administered before the incision of the scalp for localized anesthesia, and before cauterizing vessels of the scalp and skull. The remaining hole in the skull around the electrode was filled with gelatin foam (SPONGOSTAN, Søborg, Denmark), then covered with dental acrylic (Lang Dental, Jet Acrylic, Wheeling, IL, USA). Finally, an Ag/AgCl wire (5 cm long, 0.5 mm diameter) acting as a ground electrode, was inserted below the skin of the neck.
MR data acquisition and stimulation paradigms. The MRI scans were conducted in a 9.4-T 31-cm horizontal-bore magnet equipped with Agilent Direct DRIVE console (Palo Alto, CA, USA) using a quadrature radio frequency coil with full rat brain coverage. Shimming was performed inside an approximate cerebrum sized voxel (10 × 10 × 6.5 mm 3 ) using a field mapping based shimming protocol included in the Agilent VNMRJ 4.0 package. Before fMRI, high resolution anatomical images in coronal view were taken using fast spin-echo (FSE) pulse sequence: repetition time TR = 3 s, effective echo time TE = 48 ms, Echo train length = 4, matrix size = 192 × 192, field of view FOV = 32 × 32 mm 2 , slice thickness = 1 mm and number of slices = 15 from rat's AP − 4.5 mm to AP 9.5 mm, number of averages = 4. These T 2 -weighted FSE images were used to verify the electrode location and estimate the DV of the electrode tip by overlapping the MRI images with the rat brain atlas 36 ( Supplementary  Figs. 1, 2).
MB-SWIFT 58 was used for fMRI to minimize susceptibility artefacts originating from the presence of the electrodes as well as motion artefacts. MB-SWIFT is a 3D radial free induction decay (FID) based pulse sequence with virtually zero acquisition delay and high excitation and recording bandwidths. The functional contrast of MB-SWIFT has been recently investigated and attributed to blood in-flow effect providing similar results with Blood Oxygen Level Dependent (BOLD) contrast 34  where I 1,2,3 are the current amplitudes contacts 1 to 3, I 0 is the stimulation current amplitude and ∅ governs the stimulation angle. The stimulation current in each animal was set based on initial fMRI scans by driving the tripolar electrode as monopolar to identify the current level giving a non-artefactual, robust fMRI response in the hippocampus. The orientation of the electric field was set to varying angles between 0° to 315° (i.e., − 45°) with steps of 45°, for a total of 8 stimulation angles. The angles were set such that 0°/180° corresponds to the medial-lateral direction while − 90°/90° corresponds to the rostral-caudal direction, respectively (as indicated on top of Figs. 2 and 4). The order of angles was randomized across animals, as shown in Supplementary Figs. 3  and 4.
For illustration purposes, we also calculated with COMSOL 5.4 (COMSOL, Stockholm, Sweden) the voltage field distributions of OS-DBS at representative angles by considering the actual size of the 3-channel electrode, as shown in Fig. 1 and in Supplementary Fig. 1. MRI data processing and statistical analysis. MB-SWIFT images were reconstructed by gridding with three iterations FISTA algorithm 59 . The resulting data were analyzed in SPM8 (http:// www. fil. ion. ucl. ac. uk/ spm), MATLAB 2017b (The MathWorks, Inc., Natick, MA, http:// www. mathw orks. com). Prior to SPM8 analysis, functional data were first corrected for motion artefacts, then co-registered to the corresponding anatomical data and finally normalized to an animal without an electrode outside the fMRI group based on FSE images using the transformation of the anatomical data as done previously 32 . Then data was smoothed with a [1 1 1] pixel FWHM Gaussian kernel. The single-subject analysis was computed using a general linear model that consisted of a block design model convolved with a first-order gamma hemodynamic response function of 15 s duration 60 . A 1000 s cut-off high-pass filter was applied on the functional data in the temporal domain. A correction for serial auto-correlation in SPM was also applied using a first-order auto-regressive model applied to the residuals. www.nature.com/scientificreports/ At first, the level threshold for statistical significance for positive and negative activations in individual rats was set to family wise corrected (FWE) with p < 0.05. The beta maps were then used for the group-based statistical analysis by applying a one-way within-subject ANOVA model with the stimulation angle defined as a factor (8 levels, one for each angle from 0° to 315° with 45° per step) to get the maps of main effects. Maps of main effects were finally computed after applying a statistical threshold of p ≤ 0.05 (FWE). A brain mask was also applied to the group main effects.
To quantify the differences of the fMRI responses to different OS-DBS angles, we applied a linear mixed model with fixed effects for angle to the beta-values averaged in anatomically defined regions of interest (ROIs) relevant to memory and cognitive functions, ipsilateral to the stimulation site (right side), and drawn manually with Aedes (aedes.uef.fi) based on The Rat Brain in Stereotaxic Coordinates (6th Edition) 36 . The ROIs considered for OS-DBS of the EC included the hippocampus (HC) and the subiculum (Sub), i.e., areas relevant to memory and cognitive function, and areas connected to the EC, namely the perirhinal cortex (PrC), the piriform cortex (Pir), the amygdala (Amg) and the insula (Ins). The ROIs considered for OS-DBS of the MSN included the HC, the Sub, and areas connected to the MSN, namely the interpeduncular nucleus (IP), the lateral hypothalamus (LH), the mammillary bodies (MM) and supramamillary nuclei (SuM). Pair-wise comparisons of different angles were performed, and the contrast between each pair of angles was tested against a two-sided null hypothesis. Bonferroni correction was performed separately for the comparison of each angle to the other 7 angles, and level of significance was set at p = 0.05 Bonferroni corrected (i.e., p = 0.007 uncorrected). Descriptive statistics measures are indicated as Mean ± SD.
Diffusion MRI tractography and histology. The assessment of axon orientations within EC and MSN was achieved by diffusion MRI and histology as in our previous studies 30,32 . For this work, three adult male Sprague-Dawley rats outside the DBS group (300 g, Harlan Netherlands B.V., Horst, Netherlands) were used. The rats were housed in an individual cage and kept under a normal 12 h light/12 h dark cycle with constant temperature (22 ± 1 °C) and humidity (50-60%). Water and food were available ad libitum. This animal procedure was approved by the Animal Ethics Committee of the Provincial Government of Southern Finland and carried out in accordance with the guidelines of the European Community Council Directives 2010/63/EEC.
The data was pre-processed using MRtrix3 61 consisting of denoising 62 and simultaneous motion and residual eddy current induced geometric distortion correction. In order to increase anatomical contrast, the data was upsampled by a factor of two using cubic interpolation. Constrained spherical deconvolution (CSD) was used to resolve crossing-fibers by estimating the fiber orientation distribution at each voxel. The response function for white and gray matter and free water was calculated with Dhollander algorithm within MRtrix3 61 . A CSD-based whole-brain probabilistic tractography dataset of 30 million streamlines were computed. For the tractogram, iFOD2 algorithm was used 63 with a step-size of 0.037 mm, maximum angle of 45° between steps, with a minimum and maximum streamline length of 2 mm and 5 mm, respectively. The resultant tracks were filtered with spherical-deconvolution informed filtering of tractograms 64 in the regions of interest to five hundred thousand streamlines.
Coronal and horizontal sections from two rat brains stained with gold chloride for myelin 65 were used to verify the orientation of the myelinated axons in EC and MSN. Photomicrographs were taken with a light microscope (Zeiss Axio Imager2, White Plains, NY, USA) equipped with a digital camera (Zeiss Axiocam color 506, White Plains, NY, USA). The orientation of the axons was estimated using ImageJ software (1.47v, NIH, USA).

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.